The term “endotoxin” is most commonly used to refer to a lipopolysaccharide complex associated with the outer membrane of Gram-negative bacteria, although a variety of microorganisms have “endotoxin-like” components. Endotoxin is associated with a variety of negative effects on cells and tissues, including promoting cell death and triggering proinflammatory cytokines and nitric oxide. At sufficient levels in the body, endotoxin can produce “toxic shock,” a life-threatening condition. Nakagawa et al. (Nakagawa, Y. et al. “Endotoxin Contamination in Wound Dressings Made of Natural Biomaterials, J. Biomed. Mater. Res. Part B: Appl. Biomater. 66B: 347-355, 2003) demonstrated that endotoxin contamination in nine different natural wound dressings could produce fever in rabbits.
A variety of methods have been developed for removing endotoxin from proteins. These methods, however, do not lend themselves to the production of large quantities of endotoxin-free compositions. For years, complete endotoxin removal was usually achieved only with massive loss of substrate protein during the process. More recently, products have been developed for increased endotoxin removal with significant recovery of the substrate protein, but to date these products and the methods upon which they are based have not been suitable for the production of large quantities of endotoxin-free protein in a cost-effective manner. Some methods, such as that described by Naidu (U.S. Pat. No. 7,125,963) utilize a multi-step, multi-reagent approach to endotoxin removal. For some products, it is desirable to limit the use of some of these reagents, such as detergents/surfactants, which can be costly when used in large quantities. Thus such a method may be cost-prohibitive for preparation of some products.
Lactoferrin is a multi-functional protein belonging to the transferrin family of proteins. It is an 80 kDa protein, found primarily in milk and mucosal secretions. Lactoferrin binds iron, heparin, proteoglycan, DNA, oligodeoxynucleotides, and LPS (endotoxin). Two LPS binding sites have been identified in lactoferrin, the 28-34 loop region and an N-terminal four arginines (residues 2-5). The effectiveness of lactoferrin against the biological effects of endotoxins has been demonstrated. However, lactoferrin readily binds endotoxin—and it would be desirable to remove the bound endotoxin and improve the overall benefit that lactoferrin can provide. Furthermore, it would be beneficial to develop methods for removing endotoxin from large quantities of a variety of different proteins, and especially from those proteins that bind endotoxin.